CN103444531A - Rapid propagation method for tissue culture of phoebe zhennan - Google Patents
Rapid propagation method for tissue culture of phoebe zhennan Download PDFInfo
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Abstract
The invention relates to a rapid propagation method for tissue culture of phoebe zhennan. The rapid propagation method comprises the following steps: (1) selecting seeds; pre-treating the seeds and immersing the seeds in a base material for germination; after embryos are differentiated, transferring and culturing; (2) carrying out bud induction culture by taking tender coppice shoots as explants; (3) cutting the newborn tender seedlings into sections and carrying out rooting culture; (4) inoculating adventitious root buds into a proliferation culture medium and carrying out proliferation culture; (5) inoculating the adventitious root buds obtained by the proliferation culture into a rooting culture medium and carrying out rooting culture; (6) domesticating and transplanting. According to the rapid propagation method, the rooting culture of seedling stem section tissues is combined with adventitious root bud induction proliferation culture to reasonably domesticate and circularly take materials, so as to keep complete genomes of the phoebe zhennan, improve the survival rate and the quality of the phoebe zhennan and increase the growing speed of the phoebe zhennan.
Description
Technical field
The present invention relates to field of plant tissue culture technique, particularly relates to the quick-breeding method that a kind of silkwood tissue is cultivated.
Background technology
Silkwood (formal name used at school: be pH oebe zhennanS.Lee et F.N.Wei) the evergreen megaphanerophyte of Lauraceae; country's second class protection vulnerable species; for China peculiar; it is the Precious Timber Species had won fame both at home and abroad; be again that famous flower garden is viewed and admired and the urban afforestation seeds, mainly be distributed at home the ground such as West of Hubei Province, northwestern Guizhou and Sichuan.According to " guide conversant with things of the past ", nanmu has three kinds, i.e. Randia canthioides, silkwood and Shui Nan.Wherein, the most precious with silkwood, it belongs to Chinese Second Class Key Protected Plant, just endangered in the end of the Ming Dynasty.The material of silkwood has under protection against the tide, corrosion-resistant, indeformable, gentle delicate fragrance, cool in summer and warm in winter, illumination characteristics such as can sending silk silk golden light.Since ancient times, silkwood is exactly the important material of use of royal palace, minority temple building and furniture, is the rare good economic tree that integrates biodiesel, medicinal, spices and important timber, plants remarkable in economical benefits, and market prospects are wide.But because the successive dynasties felling utilizes, add that the growth rate of silkwood is slower, cause these abundant forest resources to be bordering on exhaustion.Existing silkwood woods mostly is semi-natural woods and the landscape reservation woods of the breeding of artificial organ cultivation, at temple, cottage, park, garden etc., locates that a small amount of large tree is still arranged, but the harm of sick worm is more serious, also in succession in decline.China, by years of researches, tentatively grasps the growth characteristics of silkwood and the propagation technique that tissue is cultivated thereof.The breeding of nanmu is mainly the technical staff who relies on state-owned forest farms for many years at present, utilizes the nanmu seed resource grown seedlings and cultivate.Yet the tissue culture propagation of silkwood there is not yet relevant report.
A kind of fast cuttage propagation method for silkwood as disclosed as CN101569264A, comprise fast numerous booth construction of base, with perlite, makes its matter of cuttage; Get and give birth to then or perennial lignification or the healthy and strong branch of semi-lignified, be cut into and be about 2～4cm cuttings, every cuttings band 1 leaf 1 bud; Carry out the sterilization in seedbed and the sterilization of cuttings; Process and transplant a cutting with root-growing agent or root-inducing powder; After cuttage, the water content of matrix is controlled at 60% left and right, and ambient humidity is controlled at 95% left and right, and the sunshade net bilayer of light transmittance 50% shelters from heat or light, and temperature is controlled between the 20-30 degree; Test seedling and transplanting.This scheme be take then and to be given birth to or perennial lignification or the healthy and strong branch of semi-lignified are material, and the silkwood seedling quality of turning out is not ideal enough.
The quick-breeding method that the disclosed a kind of photinia glabra tissue of CN102379241B is cultivated for another example, be to get photinia glabra stem section, is trimmed to explant, after sterilizing, induces, and obtains indefinite bud; Indefinite bud is carried out to the subculture cultivation, breed afterwards cultivation; Indefinite bud after propagation is cultivated carries out culture of rootage, obtains the seedling of taking root; The seedling hardening of taking root, being transplanted in the breeding matrix that the sterilization tissue cultivates and cultivating, and obtains the photinia glabra regeneration plant.This scheme is applicable to the tissue of photinia glabra and cultivates, because photinia glabra is rosaceous plant, different with biological tissue's characteristics and the growth needs of the silkwood of Lauraceae, Gu this scheme is unsuitable for the cultivation of silkwood.
Summary of the invention
In order to solve the problems of the technologies described above, the quick-breeding method that the object of the present invention is to provide a kind of silkwood tissue to cultivate.
The invention provides following technical scheme:
The quick-breeding method that a kind of silkwood tissue is cultivated, is characterized in that, comprises the following steps:
(1) seed selection, put into matrix after pretreatment, immersion and sprout, and after the embryo differentiation, shifts and cultivate:
Select to protect silkwood seed full, the nothing wound, hold liquid with 800 times of carbendazim and carry out pretreatment, after the root-growing agent of again seed being put into to 20ppm soaks 30min and spills, put into pearlite interstitial substance, lay with the distance of 4cm * 4cm, then water spray waters, treat seed germination, when the rootlet growth is arranged after embryo is broken up, then seed is moved on in the seedbed of having sterilized and cultivated, obtain young tender seedling and young tender coppice shoot;
(2) get after the tender seedling segment of newborn children is tamed and carry out again culture of rootage:
Get young tender seedling, flowing water rinses half an hour, be cut into about 1.5cm long, stem section with 2 joints, 0.15% mercuric chloride solution is surface sterilization 15min, sterile water wash 8 times, being inoculated in medium component is MS+BA1.0mg/L+ white sugar 2%, in the domestication bottle of 1/2 improvement MS of pH value 5.7 ± 0.1, tamed, put into again medium component and be on the root media of 1/2 improvement MS+0.6mg/L TBA+0.6mg/L NAA+0.6mg/L IAA and carry out culture of rootage, cultivation temperature is 25 ± 1 ℃, and intensity of illumination is 2500～3000LX, obtains the silkwood test-tube plantlet;
(3) getting young tender coppice shoot carries out bud as explant and induces cultivation:
With 800 times of carbendazim solutions, the tender coppice shoot of children is carried out to pretreatment, root-growing agent solution immersion treatment 15min after 3 days is put into the tender coppice shoot of children in pretreatment, rinse afterwards 30min, be placed on operating desk, be cut into the stem section with 1 to 2 internode, then with 75% Ethanol Treatment 15s, then use 0.1% mercuric chloride immersion treatment 10min, sterile water wash 8 times, in the bud domestication bottle that to be inoculated in medium component be MS+BA2.0mg/L+NAA0.1mg/L;
(4) indefinite clump bud is inoculated in proliferated culture medium and breeds cultivation:
After in step (3) bud domestication bottle, producing indefinite clump bud, be inoculated in proliferated culture medium, obtain more indefinite clump bud; Described proliferated culture medium be take modified MS medium as minimal medium, NH in the composition of described modified MS medium
4nO
3concentration reduce to 825mg/L, the proliferated culture medium composition is: improvement MS+825mg/L NH
4nO
3+ 1.0mg/L BA;
(5) will breed the indefinite clump bud cultivate obtained is inoculated in root media and carries out culture of rootage:
From step (4), propagation is cultivated in the indefinite clump bud obtained and is selected the bud of robust growth, plant height 1.8～2.2cm to cut down, be transferred in the root media described in step (3), carry out culture of rootage according to the described condition of step (3), obtain the silkwood test-tube plantlet;
(6) hardening and transplanting:
Silkwood transplantation of seedlings in root media bottle in step (2), (5) is carried out to hardening to the hardening canopy, after 7d, it is carefully taken out from bottle, clean the root medium, with 0.1% potassium permanganate immersion treatment 5min, clean, the ABT root-inducing powder that picks 25mg/L is transplanted in the matrix of getting the peat composed of rotten mosses, disinfectant ready, carry out seedling bed temperature management in canopy after transplanting, add a cover the plastic foil insulation, add a cover the rate of sheltering from heat or light 80%～90%, every 7d sprays 800 times of sterilization liquids, and described sterilization liquid is 75% carbendazim or 25% methyl Tobe, is used alternatingly.
The present invention take in described step (2), (5) (6) that to cultivate the silkwood tender seedling of children and the young tender coppice shoot that obtain be material, and the Cyclic culture of silkwood tissue is carried out in the operation of repeating step (2), (3), (4), (5), (6).
The present invention adopts improvement MS and 1/2MS medium, is conducive to the success that the silkwood tissue is cultivated.Medium is the base that during tissue is cultivated, in vitro material is depended on for existence and development, the necessary organic nutrition composition of plant growth, trace element and growth regulator, consists of.The composition of medium is that the nutrition required according to growth and development of plants designs, and not only comprises the elements such as nitrogen, phosphorus, potassium and zinc that plants needs, copper, molybdenum, iron, also comprises the organic substance of grown promotion and regulating action and plant hormone etc.Different medium, can be not just the same to the reflection of test material due to the amount difference of contained mineral salt yet.The MS medium is minimal medium commonly used, and the concentration of its mineral salt and ion is higher, is comparatively stable balance culture fluid.Because the nitrate content of MS medium is higher, so be widely used in plant organ, flower pesticide, the cultivation of cell nucleus protoplast.And the 1/2MS medium is to reduce by half by the macroelement by MS, other elements are constant, and the medium that configuration obtains.The characteristics of MS medium are mainly that inorganic salt concentration decreases, and effect and MS are roughly the same.In practical operation, normally according to the growth needs of different plants, select MS medium or 1/2MS medium.The tissue of silkwood is cultivated to be needed to control the inorganic salt concentration in medium, so the present invention carries out the domestication before culture of rootage, the differentiation rate of taking root after guaranteeing with 1/2 improvement MS.
A difficult point of silkwood group training, be how to make silkwood pseudometaplasia to transfer to the variation of indoor (outdoor) environment from outdoor (indoor) environment.The present invention adopts and induces cultivation and culture of rootage to combine, and has improved the success rate that the silkwood tissue is cultivated.As the domestication in step (2), can assist silkwood to be organized in culture of rootage afterwards and improve rooting rate, tissue be taken root and accelerate differentiation.Acclimatization and transplants in step (6) is also to make silkwood group training seedling adapt to better the process that external environment changes.
Silkwood is organized in the ability that often lacks synthetic auxin and the basic element of cell division under isolated culture condition, therefore in order to reach the effect of Promote cell's growth, division, needs as a rule to use the growth regulators such as growth hormone and the basic element of cell division simultaneously.Below by the medium component of multiplicative stage, the choice experiment of condition of culture, and the choice experiment of the medium component in the stage of taking root is set forth beneficial effect of the present invention.
Testing 1 selection of taking root the stage medium tests:
1.1 test method
The 1/2 improvement MS of take is minimal medium, adds wherein TBA, NAA and IAA; Wherein the mass concentration of TBA is set to: 0.2,0.6,1.0mg/L, the NAA(methyl α-naphthyl acetate) mass concentration is set to: 0.2,0.6,1.0mg/L, the IAA(heteroauxin) mass concentration is set to: 0.2,0.6 1.0mg/L, do trial test, repeat for 3 times, seedling in 30 bottles of every processing is added up rooting rate after 20d, observes the root growth situation.The variable concentrations combination of regulators is as shown in table 1 on the impact of culture of rootage result.
1.2 results and analysis
The impact of table 1 variable concentrations combination of regulators on the culture of rootage result
In table, data represent mean value ± standard deviation.
As can be seen from Table 1, on the medium of variable concentrations combination of regulators, rooting efficiency has difference to silkwood tissue (the indefinite clump bud that in the silkwood seedling stem section adopted in step (2), step (5), propagation obtains).When TBA, NAA, IAA concentration all are 0.6, rooting efficiency is best, and average rooting rate reaches 93%, and radical is many, and root system is long and sturdy; When TBA, NAA, IAA concentration all>during 0.6mg/L, along with the increase of concentration, rooting rate reduces, and rooting rate is slow, radical is few, root system is carefully short.In experiment, observe, culture of rootage is after 4 weeks, and silkwood seedling root differentiation rate reaches more than 40%, and root system is sturdy.Draw thus, during the silkwood culture of rootage, the optimum concentration of adding TBA, NAA, IAA is combined as: 1/2 improvement MS+0.6mg/LTBA+0.6mg/LNAA+0.6mg/LIAA can reach good rooting efficiency.Usually, the tissue-culturing rapid propagation seedling due to cylinder accumulation more hormone, or, to the Susceptible change of hormone, the small part seedling can present some residual hormone effects after the later stage root of hair, therefore, suitable hormone combinations proportioning is very important.Hormone in medium proportioning of the present invention is scientific and reasonable, is conducive to the culture of rootage of silkwood tissue.
Test the selection test of 2 multiplicative stage medium:
2.1 test method
With NH
4nO
3the concentration modified MS medium of reducing to 825mg/L be minimal medium, add respectively hormone BA(6-benayl aminopurine) 0,1.0,2.0,3.0mg/L, carry out trial test, repeat for 3 times, 20 bottles of every processing inoculations, after 30d to growth coefficient, observe the upgrowth situation of bud, and the height increment of seedlings of investigation seedling, growth coefficient etc., filter out the suitableeest hormone combination that is applicable to adventitious buds proliferation.Observed result is as shown in table 2.
2.2 results and analysis
Table 2 variable concentrations BA breeds cultivation results
In table, growth coefficient represents mean value ± standard deviation.+: growing way is general; ++: grow fine; +++growing way is excellent, without+: growing way is not obvious.
As can be seen from Table 2, when BA concentration is 1.0, indefinite clump bud growing way is best, and indefinite clump bud quantity is many, grows vigorous, and average growth coefficient reaches 3.4; The BA concentration added is 0 o'clock, and growth coefficient is minimum, and indefinite clump bud is without obvious growing way; When BA concentration>1.0, along with the increase of BA concentration, indefinite clump bud growing way reduces, and growth coefficient also reduces.Therefore, experimental result means that the optimal medium that is suitable for the indefinite clump of silkwood tissue cultivation bud propagation is: improvement MS+825mg/L NH
4nO
3+ 1.0mg/L BA, can improve the growth coefficient that the silkwood group is trained indefinite clump bud.
Test the selection test of 3 multiplicative stage condition of culture:
3.1 test method
Adopt the proliferated culture medium in step of the present invention (4), carrying out the propagation of the indefinite clump of silkwood group training bud cultivates, carry out the factorial experiments of temperature, intensity of illumination and light application time, the screening optimal culture condition, wherein temperature setting is set to 22,25,28 ℃ of these 3 processing, light application time is set to 11,13,15h, and intensity of illumination takes 1500,2500, these 3 processing of 3000LX, repeats 3 times, 30 bottles of every processing, observe the seedling upgrowth situation after 30d, record different factors and combine corresponding coefficient of differentiation, filter out the factor combination that the most applicable propagation is cultivated.Different temperature, intensity of illumination and the combination of light application time factor on the test-tube plantlet differentiation to affect result of the test as shown in table 3.
3.2 results and analysis
The different factor of table 3 combines the impact on Proliferation, Differentiation
As can be seen from Table 3, temperature, intensity of illumination and light application time factor are also very large on the Proliferation, Differentiation impact of silkwood tissue.Adopt three factor three horizontal quadrature designs, result of the test means, best with the 5th group of effect in 9 groups of tests; Coefficient of differentiation reaches 4.2; Analyzing known optimum factor from each mean value is combined as: 25 ℃ of temperature, intensity of illumination 2500LX, light application time 15h.When intensity of illumination is 2500LX and 3000LX, coefficient of differentiation is higher.The Proliferation, Differentiation of test explanation silkwood tissue needs stronger illumination, longer light application time, and moderate moisture.The propagation condition of culture that the present invention adopts meets the needs of silkwood condition of culture, is conducive to the differentiation of silkwood tissue.
4.1 test method
Adopt the identical medium of the domestication medium component front with culture of rootage (1/2 improvement MS of MS+BA1.0mg/L+ white sugar 2%), the step according to the present invention (2) is described, cultivates silkwood seedling stem section, wherein, Medium's PH Value is set to 5.4,5.5,5.6,5.7,5.8,5.9,6.0, tamed, carry out again culture of rootage, tissues observed is cultivated the situation of taking root, and adds up different pH values corresponding coefficient of differentiation and rooting rate, and the result obtained is as shown in table 4.
4.2 results and analysis
The impact of table 4pH value on the domestication of silkwood stem section and differentiation
As shown in Table 4, the pH value is larger on the coefficient of differentiation impact of silkwood seedling stem section tissue, and the pH value is 5.7 ± 1 o'clock, and the differentiation effect of taking root is better.When pH value<5.6, affect solidifying of agar, can't inoculate, unfavorable to the growth of plant; The pH value is between 5.6～5.8 the time, and medium hardness is moderate, is conducive to the growth of group training seedling, and coefficient of differentiation is high.The pH value is higher than 5.8, and coefficient of differentiation descends gradually, but coefficient of differentiation when lower than the pH value is high.Result of the test repeatedly shows, the front domestication of taking root of silkwood is best with pH value 5.7, and rooting rate and coefficient of differentiation are the highest.
The test 5 circulation tests of drawing materials
5.1 test method
Get the silkwood test-tube plantlet that obtains through culture of rootage in the present invention as test material, described in step of the present invention (2), (3), (4), (5), (6), the tender coppice shoot of children of intercepting test-tube plantlet and the stem section of test-tube plantlet, carry out the cultivation of silkwood; The tender seedling of wild silkwood children of take is the check experiment material, is under equal conditions organized culture experiment, and every processing 10 strains, repeat for 3 times, observes silkwood group training situation, the average rooting rate of statistics culture of rootage and the average survival after acclimatization and transplants.The circulation result of the test of drawing materials is as shown in table 5.
5.2 results and analysis
Rooting rate and survival rate that table 5 different tests material structure is cultivated
As shown in table 5, the test-tube plantlet stem section that adopts culture of rootage of the present invention to obtain and young tender coppice shoot be as group training material, and its rooting rate and survival rate organizes rooting rate and the survival rate of training apparently higher than take wild children tender seedling stem section and the tender coppice shoot of wild[l children as group training material.Therefore, the tender seedling of children that circulation adopts of the present invention group of training to obtain and young tender coppice shoot are group training material, can improve rooting rate and the survival rate of silkwood tissue-culturing rapid propagation.
Beneficial effect of the present invention: adopt seedling stem section to organize culture of rootage, with indefinite clump bud, inducing propagation to cultivate combines, rationally domestication, circulation is drawn materials, the complete genome group that has farthest retained silkwood, improve survival rate and the quality of silkwood, accelerated the growth rate of silkwood.Adopt the silkwood seedling of silkwood test-tube plantlet Cyclic culture virus-free, robust growth, leaf dark green, can improve the plant quality, improves survival rate; Breeding is fast, favourable to the popularization of silkwood new varieties, has solved that the new varieties material is few, the slow problem of promotion rate aborning.The silkwood tissue-culturing quick-propagation is not subject to seasonal effect, all can carry out throughout the year.Under manually operated condition, but factorial seedling growth provides seedling according to need of production for production.Acclimatization and transplants has young leaves length to grow sturdily after 40 days, now adding up transplanting survival rate is more than 80%.
The specific embodiment mode
The specific embodiment of below cultivating with the silkwood tissue is next, and the invention will be further described, to support claim of the present invention, but do not form the restriction to the claims in the present invention protection domain.
Embodiment 1
(1) seed selection, put into matrix after pretreatment, immersion and sprout, and after the embryo differentiation, shifts and cultivate:
Select to protect silkwood seed full, the nothing wound, hold liquid with 800 times of carbendazim and carry out pretreatment, after the root-growing agent of again seed being put into to 20ppm soaks 30min and spills, put into pearlite interstitial substance, lay with the distance of 4cm * 4cm, then water spray waters, treat seed germination, when the rootlet growth is arranged after embryo is broken up, then seed is moved on in the seedbed of having sterilized and cultivated, obtain young tender seedling and young tender coppice shoot;
(2) get after the tender seedling segment of newborn children is tamed and carry out again culture of rootage:
Get young tender seedling, flowing water rinses half an hour, be cut into about 1.5cm long, stem section with 2 joints, 0.15% mercuric chloride solution is surface sterilization 15min, sterile water wash 8 times, being inoculated in medium component is MS+BA1.0mg/L+ white sugar 2%, in the domestication bottle of 1/2 improvement MS of pH value 5.6, tamed, put into again medium component and be on the root media of 1/2 improvement MS+0.6mg/L TBA+0.6mg/L NAA+0.6mg/L IAA and carry out culture of rootage, cultivation temperature is 24 ℃, and intensity of illumination is 2500LX, obtains the silkwood test-tube plantlet;
(3) getting young tender coppice shoot carries out bud as explant and induces cultivation:
With 800 times of carbendazim solutions, the tender coppice shoot of children is carried out to pretreatment, root-growing agent solution immersion treatment 15min after 3 days is put into the tender coppice shoot of children in pretreatment, rinse afterwards 30min, be placed on operating desk, be cut into the stem section with 1 to 2 internode, then with 75% Ethanol Treatment 15s, then use 0.1% mercuric chloride immersion treatment 10min, sterile water wash 8 times, in the bud domestication bottle that to be inoculated in medium component be MS+BA2.0mg/L+NAA0.1mg/L;
(4) indefinite clump bud is inoculated in proliferated culture medium and breeds cultivation:
After in step (3) bud domestication bottle, producing indefinite clump bud, be inoculated in proliferated culture medium, obtain more indefinite clump bud; Described proliferated culture medium be take modified MS medium as minimal medium, NH in the composition of described modified MS medium
4nO
3concentration reduce to 825mg/L, the proliferated culture medium composition is: improvement MS+825mg/L NH
4nO
3+ 1.0mg/L BA;
(5) will breed the indefinite clump bud cultivate obtained is inoculated in root media and carries out culture of rootage:
From step (4), propagation is cultivated in the indefinite clump bud obtained and is selected the bud of robust growth, plant height 1.8cm to cut down, be transferred in the root media described in step (3), carry out culture of rootage according to the described condition of step (3), obtain the silkwood test-tube plantlet;
(6) hardening and transplanting:
Silkwood transplantation of seedlings in root media bottle in step (2), (5) is carried out to hardening to the hardening canopy, after 7d, it is carefully taken out from bottle, clean the root medium, with 0.1% potassium permanganate immersion treatment 5min, clean, the ABT root-inducing powder that picks 25mg/L is transplanted in the matrix of getting the peat composed of rotten mosses, disinfectant ready, carry out seedling bed temperature management in canopy after transplanting, add a cover the plastic foil insulation, add a cover the rate of sheltering from heat or light 80%, every 7d sprays 800 times of sterilization liquids, and described sterilization liquid is 75% carbendazim or 25% methyl Tobe, is used alternatingly.
Group training seedling is after hardening, the medium adhered to the warm water cleaning root system, be transplanted in the matrix (perlite, humus soil, liver moss, bagasse) through the potassium permanganate sterilizing, move on in greenhouse and cultivated, first use the shading screen shading after 1 month, can untie sunshade net, spray every day 3 times, 7d after transplanting, foliage-spray bactericidal liquid, spray 1 time every 7d later, and carry out the extermination of disease and insect pest.
Select the well-drained land development booth of surrounding, double-layered sunshade net in canopy, automatic sprinkling system, light-supplementing system, the wide 1.3～1.6m in seedbed, spread the humus soil of thick 25～35cm, plant 42 centimetres of plant hole specification 42 cm x 42 cm x, composite fertilizer's 220 grams of often spreading manuer in holes, mix fertilizer thoroughly during backfill table soil.For keeping root system moistening, should, with lifting with making beating, should add 3%～5% fused calcium magnesium phosphate in mud.Accomplish during transplanting that shoot root is unfolded, the seedling body is rectified, plant dark appropriateness.Silkwood seedling primary growth is slow, likes dark and damply, should select that short, irrigation and drainage of sunshine time are convenient, fertile damp soil is made ground, garden.Soil property is glutinous heavy, and impeded drainage, easily occur rotten; The antecedent soil moisture lack of water, growth of seedling is bad, easily causes and burns again.Be the silkwood seedling fast-growing phase 8～October, during this period, should strengthen the nursery water and fertilizer management, to accelerate seedling growth, improves seedling quality.Also have part plant sprouting growth November, therefore, in the final-period management of nursery, suffer freeze injury in the time of noting avoiding Overwintering Seedlings.
Embodiment 2
(1) seed selection, put into matrix after pretreatment, immersion and sprout, and after the embryo differentiation, shifts and cultivate:
Select to protect silkwood seed full, the nothing wound, hold liquid with 800 times of carbendazim and carry out pretreatment, after the root-growing agent of again seed being put into to 20ppm soaks 30min and spills, put into pearlite interstitial substance, lay with the distance of 4cm * 4cm, then water spray waters, treat seed germination, when the rootlet growth is arranged after embryo is broken up, then seed is moved on in the seedbed of having sterilized and cultivated, obtain young tender seedling and young tender coppice shoot;
(2) get after the tender seedling segment of newborn children is tamed and carry out again culture of rootage:
Get young tender seedling, flowing water rinses half an hour, be cut into about 1.5cm long, stem section with 2 joints, 0.15% mercuric chloride solution is surface sterilization 15min, sterile water wash 8 times, being inoculated in medium component is MS+BA1.0mg/L+ white sugar 2%, in the domestication bottle of 1/2 improvement MS of pH value 5.7, tamed, put into again medium component and be on the root media of 1/2 improvement MS+0.6mg/L TBA+0.6mg/L NAA+0.6mg/L IAA and carry out culture of rootage, cultivation temperature is 25 ℃, and intensity of illumination is 2800LX, obtains the silkwood test-tube plantlet;
(3) getting young tender coppice shoot carries out bud as explant and induces cultivation:
With 800 times of carbendazim solutions, the tender coppice shoot of children is carried out to pretreatment, root-growing agent solution immersion treatment 15min after 3 days is put into the tender coppice shoot of children in pretreatment, rinse afterwards 30min, be placed on operating desk, be cut into the stem section with 1 to 2 internode, then with 75% Ethanol Treatment 15s, then use 0.1% mercuric chloride immersion treatment 10min, sterile water wash 8 times, in the bud domestication bottle that to be inoculated in medium component be MS+BA2.0mg/L+NAA0.1mg/L;
(4) indefinite clump bud is inoculated in proliferated culture medium and breeds cultivation:
After in step (3) bud domestication bottle, producing indefinite clump bud, be inoculated in proliferated culture medium, obtain more indefinite clump bud; Described proliferated culture medium be take modified MS medium as minimal medium, NH in the composition of described modified MS medium
4nO
3concentration reduce to 825mg/L, the proliferated culture medium composition is: improvement MS+825mg/L NH
4nO
3+ 1.0mg/L BA;
(5) will breed the indefinite clump bud cultivate obtained is inoculated in root media and carries out culture of rootage:
From step (4), propagation is cultivated in the indefinite clump bud obtained and is selected the bud of robust growth, plant height 2.0cm to cut down, be transferred in the root media described in step (3), carry out culture of rootage according to the described condition of step (3), obtain the silkwood test-tube plantlet;
(6) hardening and transplanting:
Silkwood transplantation of seedlings in root media bottle in step (2), (5) is carried out to hardening to the hardening canopy, after 7d, it is carefully taken out from bottle, clean the root medium, with 0.1% potassium permanganate immersion treatment 5min, clean, the ABT root-inducing powder that picks 25mg/L is transplanted in the matrix of getting the peat composed of rotten mosses, disinfectant ready, carry out seedling bed temperature management in canopy after transplanting, add a cover the plastic foil insulation, add a cover the rate of sheltering from heat or light 85%, every 7d sprays 800 times of sterilization liquids, and described sterilization liquid is 75% carbendazim or 25% methyl Tobe, is used alternatingly.
Take in described step (2), (5) (6) that to cultivate the silkwood tender seedling of children and the young tender coppice shoot that obtain be material, the operation of repeating step (2), (3), (4), (5), (6), carry out the Cyclic culture of silkwood tissue.
Embodiment 3
(1) seed selection, put into matrix after pretreatment, immersion and sprout, and after the embryo differentiation, shifts and cultivate:
Select to protect silkwood seed full, the nothing wound, hold liquid with 800 times of carbendazim and carry out pretreatment, after the root-growing agent of again seed being put into to 20ppm soaks 30min and spills, put into pearlite interstitial substance, lay with the distance of 4cm * 4cm, then water spray waters, treat seed germination, when the rootlet growth is arranged after embryo is broken up, then seed is moved on in the seedbed of having sterilized and cultivated, obtain young tender seedling and young tender coppice shoot;
(2) get after the tender seedling segment of newborn children is tamed and carry out again culture of rootage:
Get young tender seedling, flowing water rinses half an hour, be cut into about 1.5cm long, stem section with 2 joints, 0.15% mercuric chloride solution is surface sterilization 15min, sterile water wash 8 times, being inoculated in medium component is MS+BA1.0mg/L+ white sugar 2%, in the domestication bottle of 1/2 improvement MS of pH value 5.8, tamed, put into again medium component and be on the root media of 1/2 improvement MS+0.6mg/L TBA+0.6mg/L NAA+0.6mg/L IAA and carry out culture of rootage, cultivation temperature is 26 ℃, and intensity of illumination is 3000LX, obtains the silkwood test-tube plantlet;
(3) getting young tender coppice shoot carries out bud as explant and induces cultivation:
With 800 times of carbendazim solutions, the tender coppice shoot of children is carried out to pretreatment, root-growing agent solution immersion treatment 15min after 3 days is put into the tender coppice shoot of children in pretreatment, rinse afterwards 30min, be placed on operating desk, be cut into the stem section with 1 to 2 internode, then with 75% Ethanol Treatment 15s, then use 0.1% mercuric chloride immersion treatment 10min, sterile water wash 8 times, in the bud domestication bottle that to be inoculated in medium component be MS+BA2.0mg/L+NAA0.1mg/L;
(4) indefinite clump bud is inoculated in proliferated culture medium and breeds cultivation:
After in step (3) bud domestication bottle, producing indefinite clump bud, be inoculated in proliferated culture medium, obtain more indefinite clump bud; Described proliferated culture medium be take modified MS medium as minimal medium, NH in the composition of described modified MS medium
4nO
3concentration reduce to 825mg/L, the proliferated culture medium composition is: improvement MS+825mg/L NH
4nO
3+ 1.0mg/L BA;
(5) will breed the indefinite clump bud cultivate obtained is inoculated in root media and carries out culture of rootage:
From step (4), propagation is cultivated in the indefinite clump bud obtained and is selected the bud of robust growth, plant height 2.2cm to cut down, be transferred in the root media described in step (3), carry out culture of rootage according to the described condition of step (3), obtain the silkwood test-tube plantlet;
(6) hardening and transplanting:
Silkwood transplantation of seedlings in root media bottle in step (2), (5) is carried out to hardening to the hardening canopy, after 7d, it is carefully taken out from bottle, clean the root medium, with 0.1% potassium permanganate immersion treatment 5min, clean, the ABT root-inducing powder that picks 25mg/L is transplanted in the matrix of getting the peat composed of rotten mosses, disinfectant ready, carry out seedling bed temperature management in canopy after transplanting, add a cover the plastic foil insulation, add a cover the rate of sheltering from heat or light 90%, every 7d sprays 800 times of sterilization liquids, and described sterilization liquid is 75% carbendazim or 25% methyl Tobe, is used alternatingly.
Take in described step (2), (5) (6) that to cultivate the silkwood tender seedling of children and the young tender coppice shoot that obtain be material, the operation of repeating step (2), (3), (4), (5), (6), carry out the Cyclic culture of silkwood tissue.
Claims (2)
1. the quick-breeding method that the silkwood tissue is cultivated, is characterized in that, comprises the following steps:
(1) seed selection, put into matrix after pretreatment, immersion and sprout, and after the embryo differentiation, shifts and cultivate:
Select to protect silkwood seed full, the nothing wound, hold liquid with 800 times of carbendazim and carry out pretreatment, after the root-growing agent of again seed being put into to 20ppm soaks 30min and spills, put into pearlite interstitial substance, lay with the distance of 4cm * 4cm, then water spray waters, treat seed germination, when the rootlet growth is arranged after embryo is broken up, then seed is moved on in the seedbed of having sterilized and cultivated, obtain young tender seedling and young tender coppice shoot;
(2) get after the tender seedling segment of newborn children is tamed and carry out again culture of rootage:
Get young tender seedling, flowing water rinses half an hour, is cut into about 1.5cm long, with the stem section of 2 joints, 0.15% mercuric chloride solution is surface sterilization 15min, sterile water wash 8 times, being inoculated in medium component is MS+BA1.0mg/L+ white sugar 2%, in the domestication bottle of 1/2 improvement MS of pH value 5.7 ± 0.1, is tamed, carry out culture of rootage on the root media that to put into medium component be MS+NAA0.6mg/L again, cultivation temperature is 25 ± 1 ℃, and intensity of illumination is 2500～3000LX, obtains the silkwood test-tube plantlet;
(3) getting young tender coppice shoot carries out bud as explant and induces cultivation:
With 800 times of carbendazim solutions, the tender coppice shoot of children is carried out to pretreatment, root-growing agent solution immersion treatment 15min after 3 days is put into the tender coppice shoot of children in pretreatment, rinse afterwards 30min, be placed on operating desk, be cut into the stem section with 1 to 2 internode, then with 75% Ethanol Treatment 15s, then use 0.1% mercuric chloride immersion treatment 10min, sterile water wash 8 times, in the bud domestication bottle that to be inoculated in medium component be MS+BA2.0mg/L+NAA0.1mg/L;
(4) indefinite clump bud is inoculated in proliferated culture medium and breeds cultivation:
After in step (3) bud domestication bottle, producing indefinite clump bud, be inoculated in proliferated culture medium, obtain more indefinite clump bud; Described proliferated culture medium be take modified MS medium as minimal medium, NH in the composition of described modified MS medium
4nO
3concentration reduce to 825mg/L;
(5) will breed the indefinite clump bud cultivate obtained is inoculated in root media and carries out culture of rootage:
From step (4), propagation is cultivated in the indefinite clump bud obtained and is selected the bud of robust growth, plant height 1.8～2.2cm to cut down, be transferred in the root media described in step (3), carry out culture of rootage according to the described condition of step (3), obtain the silkwood test-tube plantlet;
(6) hardening and transplanting:
Silkwood transplantation of seedlings in root media bottle in step (2), (5) is carried out to hardening to the hardening canopy, after 7d, it is carefully taken out from bottle, clean the root medium, with 0.1% potassium permanganate immersion treatment 5min, clean, the ABT root-inducing powder that picks 25mg/L is transplanted in the matrix of getting the peat composed of rotten mosses, disinfectant ready, carry out seedling bed temperature management in canopy after transplanting, add a cover the plastic foil insulation, add a cover the rate of sheltering from heat or light 80%～90%, every 7d sprays 800 times of sterilization liquids, and described sterilization liquid is 75% carbendazim or 25% methyl Tobe, is used alternatingly.
2. preparation method according to claim 1, it is characterized in that, take in described step (2), (5) (6) that to cultivate the silkwood tender seedling of children and the young tender coppice shoot that obtain be material, the operation of repeating step (2), (3), (4), (5), (6), carry out the Cyclic culture of silkwood tissue.
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| CN104620828A (en) * | 2015-02-14 | 2015-05-20 | 周琪 | Method of cultivating landscaping trees by wild-to-artificial cultivation of Choerospondias axillaris |
| CN106212288A (en) * | 2016-08-24 | 2016-12-14 | 华南农业大学 | A kind of tissue culture propagation method of Machilus pauhoi |
| CN106212288B (en) * | 2016-08-24 | 2018-07-31 | 华南农业大学 | A kind of tissue culture propagation method of Machilus pauhoi |
| CN106386478A (en) * | 2016-08-28 | 2017-02-15 | 李志勇 | Phoebe zhennan tissue culture rapid breeding method |
| CN106508345A (en) * | 2016-10-20 | 2017-03-22 | 江苏省林业科学研究院 | Secondary cutting propagation method for quercus nuttallii |
| CN106489736A (en) * | 2016-11-03 | 2017-03-15 | 宜宾云辰乔木园林有限责任公司 | A kind of method for quickly breeding of hardwood nanmu |
| CN108566795A (en) * | 2017-12-15 | 2018-09-25 | 江苏农牧科技职业学院 | A method of releasing purple nanmu seed dormancy |
| CN109302986A (en) * | 2018-11-16 | 2019-02-05 | 湖北民族学院 | A kind of method of nanmu select tree tissue cultures childrenization |
| CN112772416A (en) * | 2021-01-28 | 2021-05-11 | 宜宾金铁红林业科技有限公司 | Tissue culture seedling raising method for phyllostachys microphylla |
| CN112772416B (en) * | 2021-01-28 | 2022-08-12 | 苏州金铁红林业科技有限公司 | Tissue culture seedling raising method for phyllostachys microphylla |
| CN114651726A (en) * | 2022-05-05 | 2022-06-24 | 贵州大学 | Method for culturing nanmu seed embryo aseptic seedlings |
| CN114651726B (en) * | 2022-05-05 | 2023-06-20 | 贵州大学 | Method for culturing aseptic seedlings of phoebe embryo |
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